Anti-rho GTPase conformational single domain antibodies and uses thereof

ABSTRACT

The present invention relates to active form specific anti-Rho GTPase conformational single domain antibodies and their uses in particular in the therapeutic and diagnostic fields. In particular, the present invention relates to a single domain antibody wherein the amino acid sequences of CDR1-IMGT, CDR2-IMGT and CDR3-IMGT have at least 90% of identity with the amino acid sequences of the CDR1-IMGT, CDR2-IMGT and CDR3-IMGT of the H12, B6, 4P75, 4SP1, 4SNP36, 4SNP61, 5SP10, 5SP11, 5SP58, 5SNP47, 5SNP48, 5SNP65, B20, B15, B5, B71, E3, A6, G12, NB61, 212B, 111B or 404F (hs2dAb) single domain antibody which are defined in Table B.

FIELD OF THE INVENTION

The present invention relates to anti-Rho GTPase conformational singledomain antibodies and their uses thereof in particular in thetherapeutic and diagnostic fields.

BACKGROUND OF THE INVENTION

Rho GTPases belong to the family of the 20 small GTPases homologous toRas which are largely considered as undrugable proteins. Theirphysiologic activity resides more in a conformational change of theswitch I and switch II highly conserved domains than in their guaninenucleotide triphosphate hydrolase very slow catalytic activity. Small Gproteins are therefore molecular switch that cycle between an inactiveGDP bound state an active GTP bound conformation. The Rho subfamily,which contains RhoA, RhoB and RhoC that share more than 85% sequenceidentities are pleiotropic proteins involved in a wide range of majorcell processes, including the control of actomyosin cytoskeleton, celladhesion, cytokinesis, cell migration, stress response as well as cellsurvival or apoptosis. These GTPases are post-translationally modifiedby addition of a carboxy terminal isoprenoid group necessary for theiranchorage to cellular membranes, where they can be activated uponvarious stimuli by an exchange of nucleotide catalyzed by Guaninenucleotide Exchange Factors (GEF). Activation step is thencounterbalanced by GTP hydrolyses which is enhanced by GTPase ActivatingProteins (GAPs). Therefore the cellular pool of activated Rho ismaintained at the cellular membrane to limited pool. The major fractionis extracted from the membrane and sequestered in the cytoplasm byGuanine nucleotide Dissociation Inhibitors (GDI). These large proteinsinteract by their amino terminal part with the switch domains of Rho,thus preventing the release of the GDP, and also by their carboxyterminal part to the isoprenyl group, shielding this hydrophobic moietyin order to maintain the Rho proteins soluble while excluded frommembranes.

Together these regulators maintain the largest fraction of Rho Proteinsinactive in the cell, often as much as 95%, in order to quickly activatea very small population that will interact with effector proteins toinitiate cellular transduction pathways. Moreover study of the crosstalkbetween the 3 GDI and all interacting Rho revealed that overexpressionof a single Rho can induce artificially displacement and degradation ofothers and impair signaling pathways not directly controlled by thetransgene. This critical point highlights the complexity of targetingindividual Rho in a selective manner.

The peculiar RhoB seems to be involved in different cellular functionsand regulations than its closest homologs RhoA and RhoC. RhoB can bepalmitoylated and either farnesylated or geranylgeranylated, prenylationwhich define localization to the plasma membrane or the endosomerespectively. Some main RhoB functions in intracellular trafficking andadhesion have been characterized using conventional molecular tools suchas overexpression of wild type or mutants or genetic knock down by RNAinterference. Other functions have been connected to RhoB geneexpression as an immediate early response to cytokines or growth factorsas well as DNA damaging agent or radiation. In addition, RhoB playsparadoxal roles in cancer progression. RhoB can alter tumor formationand is often down regulated in head and neck or lung cancers.

Nevertheless RhoB is also promoting tumor angiogenesis and protectingfrom apoptosis in cells with genomic instability. There are now clearevidences that RhoB exert pleiotropic functions which are cells andcontext dependent. Previous studies targeted RhoB at the genetic levelby overexpression, RNAi or gene knock out in mice. However these methodsaltered RhoB functions in a global way, knocking down all RhoBactivities in cells, but mostly altering both the GDP bound majorfraction which can induce imbalances in the GDI-Rho interactions and theminor GTP bound active pool. To decipher RhoB function withoutinterfering with other Rho activities, it would be necessary to targetRhoB at the protein level. Albeit there are no small molecule inhibitorstargeting Rho GTPases, the C3 exoenzyme from Claustridium botulinum orBacillus cereus are natural inhibitors that induce ADP-ribosylation ofRho, preventing their activation by GEF and further increasing thebinding of free Rho to GDI. Actually expression of C3 gene in eukaryoticcells or incubation with cell permeable tat-C3 has been successfullyused to alter globally the function of all 3 Rho, leading to a strongphenotype of actin fiber loss and cell rounding. Several other bacterialtoxin target as well Rho proteins. Nevertheless all these toxins lackspecificity because they do not discriminate between RhoA, RhoB or RhoCand mostly do not block directly the activated form of Rho.

In some previous studies, the selection of recombinant single chainantibodies from phage display libraries was established in order toidentify binding molecules selective to the active GTP bound state ofRho proteins. Actually recombinant antibody selected from large displaylibraries have been used in many biotechnological or biomedicalapplications. Although most of recombinant antibodies require forstability the canonical disulfide bond within the VH or VL variabledomains, some peculiar intracellular antibodies, referred asintrabodies, remains stable in the reducing environment. Thus, dependingon the antibody format, the scaffold properties and the librarydiversities, some rare recombinant antibodies have been reported to befunctional while expressed in the cytosol of eukaryotic cells (Tanaka,T., Williams, R. L. & Rabbitts, T. H. Tumour prevention by a singleantibody domain targeting the interaction of signal transductionproteins with RAS. EMBO J 26, 3250-3259, doi:7601744 [pii]10.1038/sj.emboj.7601744 (2007); Nizak, C. et al. Recombinant antibodiesto the small GTPase Rab6 as conformation sensors. Science 300, 984-987,doi:10.1126/science.1083911 (2003).; Meli, G., Visintin, M.,Cannistraci, I. & Cattaneo, A. Direct in vivo intracellular selection ofconformation-sensitive antibody domains targeting Alzheimer'samyloid-beta oligomers. J Mol Biol 387, 584-606,doi:10.1016/j.jmb.2009.01.061 (2009)). The first active Rhoconformational single chain variable fragment (scFv), named scFvC1,recognized in vitro in biochemical assays the GTP bound state of all 3Rho (Goffinet, M. et al. Identification of a GTP-bound Rho specific scFvmolecular sensor by phage display selection. BMC Biotechnol 8, 34,doi:1472-6750-8-34 [pii] 10.1186/1472-6750-8-34 (2008).). A molecularevolution of scFvC1 led to the identification of the scFvF7, a higheraffinity pan active Rho binder than scFvC1, as well as the scFvE3 thatpreferentially recognizes active RhoB but is not functional as anintrabody.

SUMMARY OF THE INVENTION

The present invention relates to active form specific anti-Rho GTPaseconformational single domain antibodies and their uses in particular inthe therapeutic and diagnostic fields. In particular, the presentinvention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

The inventors produced a fully synthetic library of humanizednanobodies, common name of single domain VH from camelidae. This novelphage display library, based on a unique scaffold optimized forstability, allowed the systematic selection of highly functional bindingmolecules, which correspond to humanized synthetic single domainantibodies (hs2dAb). The inventors thus especially selected towardsvarious targets several functional intrabodies, among which oneconformational sensor of the active GTP bound state of Rho proteins, aclone referred as H12 hs2dAb. They demonstrated that the H12 hs2dAb is apan Rho binder that do not discriminate between the Rho subfamilymembers RhoA, RhoB and RhoC, and also bond members of the close Rac1family of Ras homologous small G proteins. The inventors furtherisolated by using more competitive selection scheme several active Rhoselective hs2dAb molecules with more specificity towards the Rhosubfamily, namely that do not recognize Rac1 or other small G proteins.They demonstrated that some of these hs2dAb had sub-nanomolar affinitiestowards recombinant constitutively active L63 mutant of Rhos.Intracellular expression of these molecules lead to a clearreorganization of actin cytoskeleton, suggesting that they act as strongintracellular inhibitor of Rho signaling in cultured cells. Then theinventors applied the same strategy to select single domain antibodiesspecific that recognized RhoB in its GTP bound state. In particular,they established a visual in-cell screening of functionalizedintrabodies that can induce active RhoB protein knock down. The proteinknock down was based on the use of an Fbox domain genetically fused tothe selected single domain antibodies, which induce ubiquitination ofthe bound target and subsequent proteasome dependent degradation. A dualchallenge was to identify an intrabody which can physiologicallydiscriminate RhoB from the closest homologs while being selective of theactive GTP-bound state. Using cell lines expressing various Rho mutants,the inventors succeeded in selecting a robust genetically encoded RhoBinhibitor selective of its active state and demonstrated the efficiencyof this unique tool to knock down endogenous RhoB active fraction, notonly depleting its basal activity but also blocking its cellularactivation after growth factor treatment. Furthermore, in a proof ofprinciple study the inventors demonstrated that subtle RhoB activityknock down did not displace the whole cellular fraction of RhoB butinduced similar phenotype as RNAi on human bronchial epithelial cellsmigration and invasion. Accordingly, the present invention relates toanti-Rho GTPase conformational single domain antibodies and their usesin particular in the therapeutic and diagnostic fields.

As used herein the term “Rho-GTPase” has its general meaning in the artand refers to the Rho (ras homology) family of small molecular weightguanosine triphosphatases Rho GTPases are molecular switches thatcontrol signaling pathways regulating cytoskeleton organization, geneexpression, cell cycle progression, cell motility and other cellularprocesses (Cell Communication and Signaling, 2010, 8, 23). Rho familyGTPases are important signaling proteins that control diverse cellularfunctions related to cancer development, including actin cytoskeletonorganization, transcription regulation, cell cycle progression,apoptosis, vesicle trafficking, and cell-to-cell andcell-to-extracellular matrix adhesions (Cell Communication andSignaling, 2010, 8 (23), 1-14; Genes Dev., 1997, 1 1, 2295-2322). Inparticular, Rho-GTPase includes RhoA, RhoB and RhoC.

The single domain antibodies generated by the inventors are specific forat least one Rho-GTPase, and more particularly for only one Rho-GTPase(e.g. the“B6” hs2dAb is specific for RhoB). However some antibodies ofthe present invention are able to interact with several Rho-GTPases(e.g. the H12 hs2dAb has affinity for RhoA, RhoB and RhoC as well asRac1). Accordingly, the single domain antibodies of the presentinvention are characterized by one or more functional properties suchthat they are humanized, they have a specific affinity for oneRho-GTPase or for several Rho-GTPases, they are specific for oneactivated form of the Rho-GTPase (i.e. conformational), they are able toinhibit the activated form of the Rho-GTPase, they are highly stablesingle domain antibodies, they present high affinity, and they areactive in the intracellular environment.

As used herein the term “single domain antibody” has its general meaningin the art and refers to the single heavy chain variable domain ofantibodies of the type that can be found in Camelid mammals which arenaturally devoid of light chains. Such single domain antibody are also“Nanobody®”. For a general description of (single) domain antibodies,reference is also made to the prior art cited above, as well as to EP 0368 684, Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), Holt etal., Trends Biotechnol., 2003, 21(11):484-490; and WO 06/030220, WO06/003388. The amino acid sequence and structure of a single domainantibody can be considered to be comprised of four framework regions or“FRs” which are referred to in the art and herein as “Framework region1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3”or “FR3”; and as “Framework region 4” or “FR4” respectively; whichframework regions are interrupted by three complementary determiningregions or “CDRs”, which are referred to in the art as “ComplementarityDetermining Region for “CDR1”; as “Complementarity Determining Region 2”or “CDR2” and as “Complementarity Determining Region 3” or “CDR3”,respectively. Accordingly, the single domain antibody can be defined asan amino acid sequence with the general structure:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to frameworkregions 1 to 4 respectively, and in which CDR1 to CDR3 refer to thecomplementarity determining regions 1 to 3. In the context of theinvention, the amino acid residues of the single domain antibody arenumbered according to the general numbering for VH domains given by theIMGT numbering system (Lefranc M.-P., “Unique database numbering systemfor immunogenetic analysis” Immunology Today, 18, 509 (1997)). The IMGTunique numbering has been defined to compare the variable domainswhatever the antigen receptor, the chain type, or the species (LefrancM.-P., “Unique database numbering system for immunogenetic analysis”Immunology Today, 18, 509 (1997); Lefranc M.-P., “The IMGT uniquenumbering for Immunoglobulins, T cell receptors and Ig-like domains” TheImmunologist, 7, 132-136 (1999).; Lefranc, M.-P., Pommie, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, G., “IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains” Dev. Comp.Immunol., 27, 55-77 (2003).). In the IMGT unique numbering, theconserved amino acids always have the same position, for instancecysteine 23, tryptophan 41, hydrophobic amino acid 89, cysteine 104,phenylalanine or tryptophan 118. The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths become crucial information.Gaps in the CDR1-IMGT and CDR2-IMGT (less than 12 and 10 amino acidlong, respectively) are put at the top of the CDR-IMGT loops. Forinstance, when the length of CDR1-IMGT is 7 amino acids, it comprisesthe positions 27, 28, 29, 30, 36, 37 and 38. When the length ofCDR2-IMGT is 7 amino acids, it comprises the positions 56, 57, 58, 59,63, 64, and 65. The basic length of a rearranged CDR3-IMGT is 13 aminoacids (positions 105 to 117), which corresponds to a JUNCTION of 15amino acids (2nd-CYS 104 to J-TRP or J-PHE 118). This length andcorresponding numbering were chosen since they are convenient to use.Indeed, 80% of the IG and TR rearranged sequences in IMGT/LIGM-DB have aCDR3-IMGT length less than or equal to 13 amino acids. If the CDR3-IMGTlength is less than 13 amino acids, gaps are created from the top of theloop, in the following order 111, 112, 110, 113, 109, 114, etc.Accordingly, when the length of CDR3-IMGT is 9 amino acids, it comprisesthe positions 105; 106; 107; 108; 109; 114; 115; 116; and 117. Whenlength of CDR3-IMGT is 9 amino acids, it comprises the positions 105;106; 107; 108; 109; 110; 112; 113; 114; 115; 116; and 117. If theCDR3-IMGT length is more than 13 amino acids, additional positions arecreated between positions 111 and 112 at the top of the CDR3-IMGT loopin the following order 112.1, 111.1, 112.2, 111.2, 112.3, 111.3, etc.Accordingly when the length of CDR3-IMGT is 15 amino acids, it comprisesthe additional positions 111.1 and 112.1.

All the single domain antibodies (hs2dAb) generated by the inventors arecharacterized by the same frameworks regions FR1-FR4 as described inTable A and comprises a CDR1-IMGT and a CDR2-IMGT having a length of 7amino acids and a CDR3-IMGT having a length of 9, 12 or 15 amino acidsas described in Table B.

TABLE A frameworks regions FR1-FR4 (IMGT)of the single domain antibodies (hs2dAb) generated by the inventors:Framework region Sequence FR1 VQLQASGGGFVQPGGSLRLSCAASG (SEQ ID NO: 1)FR2 MGWFRQAPGKEREFVSAISS (SEQ ID NO: 2) FR3 YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA (SEQ ID NO: 3) FR4 YWGQGTQVTVSS (SEQ ID NO: 4)

TABLE B CDR-IMGT regions of the single domainantibodies (hs2dAb) generated by the inventors: hs2dAb CDR1-IMGTCDR2-IMGT CDR3-IMGT H12 DGSRIYA WEQDWEH AFMTPH (SEQ ID (SEQ ID RNLTSMNO: 5) NO: 6) (SEQ ID NO: 7) 4P75 RYSAWDG SQHDLEE ATIRT (SEQ ID (SEQ IDGWAD NO: 8) NO: 9) (SEQ ID NO: 10) 4SP1 DTSDGYI EYNSQSE QSFNEVW (SEQ ID(SEQ ID KMPNKFPH NO: 11) NO: 12) (SEQ ID NO: 13) 4SNP36 TSWKDYT EGPGAQYYSSWQ (SEQ ID (SEQ PYVS NO: 14) ID NO: 15) (SEQ ID NO: 16) 4SNP61FTSTSTV SAHTMDT YCAPAPM (SEQ ID (SEQ LGQMITQ NO: 17) ID NO: 18) PALP(SEQ ID NO: 19) 5SP10 RFWRRYT GTSDWT PPHFS (SEQ ID (SEQ ID GAAI NO: 20)NO: 21) (SEQ ID NO: 22) 5SP11 AGWRAEA SDGDHTI IMQTQMR (SEQ ID (SEQRTSDYRF NO: 23) ID NO: 24) (SEQ ID NO: 25) 5SP58 DTFSDDV DWPTTQSYCAQANGD (SEQ (SEQ HSYPLWKY ID NO: 26) ID NO: 27) GNM (SEQ ID NO: 28)5SNP47 RTSRFYS FNSDYFL AWWYRY (SEQ ID (SEQ ID TEGMTM NO: 29) NO: 30)(SEQ ID NO: 31) 5SNP48 TSWFTEV (SEQ GLHDVGT ALDKWYTK ID NO: 32) (SEQ IDAMDARKD NO: 33) (SEQ ID NO: 34) 5SNP65 ATYEGEA (SEQ SYPSVIS YWVNHEID NO: 35) (SEQ ID GTIREI NO: 36) (SEQ ID NO: 37 B6 YGSTIET RAPGPSQPINNRTMQ (SEQ ID (SEQ ID DSMFLWN NO: 38) NO: 39) (SEQ ID NO: 40) B20TTSFWYT (SEQ WRFNTTT 1PRYSLDA ID NO: 41) (SEQ ID VPHRAST NO: 42) (SEQ IDNO: 43) B15 SYSRGET(SEQ DTHNYET ASPQFHK ID NO: 44) (SEQ ID IMKGSQVNO: 45) G (SEQ ID NO: 46) B5 ATSGGTV RSQTKAT PMEHEAL (SEQ ID (SEQ IDKQHPL NO: 47) NO: 48) (SEQ ID NO: 49) B71 DGSDGDV RYPGRSP ARWISRK(SEQ ID (SEQ ID WYTTPFQG NO: 50) NO: 51) (SEQ ID NO: 52) E3 STYETYAASPTIEG TWSKM (SEQ ID (SEQ ID GISI NO: 53) NO: 54) (SEQ ID NO: 55) A6DTWDQYV RSGTH PLTHQW (SEQ ID GI MGRTFP NO: 56) (SEQ ID (SEQ ID NO: 57)NO: 58) G12 RTSGWYA SRASSQE VWMKM (SEQ ID (SEQ ID GIEI NO: 59) NO: 60)(SEQ ID NO: 61) NB61 TTWFNEV GSTSWAE RMSFMR (SEQ ID (SEQ ID AGRTPMNO: 81) NO: 82) TPM (SEQ ID NO: 83) 212B DTWWSSA FYPTEYT WIAWGP (SEQ ID(SEQ ID WMRTSW NO: 84) NO: 85) (SEQ ID NO: 86) 111B GTSKQYG RQEGETIYRHVW (SEQ ID (SEQ ID PYPE NO: 87) NO: 88) (SEQ ID NO: 89) 404F RTSKNYAWTTNQDT IWDKR (SEQ ID (SEQ ID EISI NO: 90) NO: 91) (SEQ ID NO: 92)

The antibodies (hs2dAb) are characterized by the sequences of Table C:

TABLE C sequences of the single domain antibodies(hs2dAb) generated by the inventors: hs2dAb Sequence H12VQLQASGGGFVQPGGSLRLS CAASGDGSRIYAMGWFRQAP GKEREFVSAISWEQDWEHYYADSVKGRFTISRDNSKNTVY LQMNSLRAEDTATYYCAAFM TPHRNLTSMYWGQGTQVTVSS (SEQ ID NO: 62) 4P75 VQLQASGGGFVQPGGSLRLSC AASGRYSAWDGMGWFRQAPGKEREFVSAISSQHDLEEYYADS VKGRTISRDNSKNTVYLQMNS LRAEDTATYYCAATIRTGWADYWGQGTQVTVSS (SEQ ID NO: 63) 4SP1 VQLQASGGGFVQPGGSLRLSCAASGDTSDGYIMGWFRQAPGK EREFVSAISEYNSQSEYYADS VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAQSFNEVWK MPNKFPHYWGQGTQVTVSS (SEQ ID NO: 64) 4SNP36VQLQASGGGFVQPGGSLRLSCA ASGTSWKDYTMGWFRQAPGKER EFVSAISEGPGAQYYYADSVKGRFTISRDNSKNVYLQMNSLRAE DTATYYCAYSSWQPYVSYWGQG TQVTVSS (SEQ ID NO: 65)4SNP61 VQLQASGGGFVQPGGSLRLSCA ASGFTSTSTVMGWFRQAPGKEREFVSAISSAHTMDTYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCAPAPMLGQMITQPALPYWGQGTQVTVSS (SEQ ID NO: 66) 5SP10 VQLQASGGGFVQPGGSLRLSCAASGRFWRRYTMGWFRQAPGK EREFVSAISGTSDWTYYADSV KGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAPPHFSGAAI YWGQGTQVTVSS (SEQ ID NO: 67) 5SP11VQLQASGGGFVQPGGSLRLSCA ASGAGWRAEAMGWFRQAPGKER EFVSAISSDGDHTIYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTATYYCAIMQTQMRRTSDYR FYWGQGTQVTVSS(SEQ ID NO: 68) 5SP58 VQLQASGGGFVQPGGSLRLSCA ASGDTFSDDVMGWFRQAPGKEREFVSAISDWPTTQSYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCAQANGDHSYPLWKYGNMYWGQGTQVTVSS (SEQ ID NO: 69) 5SNP47 VQLQASGGGFVQPGGSLRLSCAASGRTSRFYSMGWFRQAPGKER EFVSAISFNSDYFLYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTATYYCAAWWYRYTEGMTMY WGQGTQVTVSS (SEQ ID NO: 70) 5SNP48VQLQASGGGFVQPGGSLRLSCA ASGTSWFTEVMGWFRQAPGKER EFVSAISGLHDVGTYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTATYYCAALDKWYTKAMDAR KDYWGQGTQVTVSS(SEQ ID NO: 71) 5SNP65 VQLQASGGGFVQPGGSLRLSCA ASGATYEGEAMGWFRQAPGKEREFVSAISSYPSVISYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCAYWVNHEGTIREIYWGQGTQVTVSS (SEQ ID NO: 72) B6 VQLQASGGGFVQPGGSLRLSCAASGYGSTIETMGWFRQAPGKER EFVSAISRAPGPSQYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTATYYCAPINNRTMQDSMFL WNYWGQGTQVTVSS (SEQ ID NO: 73) B20VQLQASGGGFVQPGGSLRLSCA ASGTTSFWYTMGWFRQAPGKER EFVSAISWRFNTTTYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTATYYCAIPRYSLDAVPHRA STYWGQGTQVTVSS(SEQ ID NO: 74) B15 VQLQASGGGFVQPGGSLRLSCA ASGSYSRGETMGWFRQAPGKEREFVSAISDTHNYETYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCAASPQFHKIMKGSQVGYWGQGTQVTVSS (SEQ ID NO: 75) B5 VQLQASGGGFVQPGGSLRLSCAASGATSGGTVMGWFRQAPGKER EFVSAISRSQTKATYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTATYYCAPMEHEALKQHPLY WGQGTQVTVSS (SEQ ID NO: 76) B71VQLQASGGGFVQPGGSLRLSCA ASGDGSDGDVMGWFRQAPGKER EFVSAISRYPGRSPYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTATYYCAARWISRKWYTTPF QGYWGQGTQVTVSS(SEQ ID NO: 77) E3 VQLQASGGGFVQPGGSLRLSCA ASGSTYETYAMGWFRQAPGKEREFVSAISASPTIEGYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCATWSKMGISIYWGQGTQVTVSS  (SEQ ID NO: 78) A6 VQLQASGGGFVQPGGSLRLSCAASGDTWDQYVMGWFRQAPGKER EFVSAISRSGTHGIYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTATYYCAPLTHQWMGRTFPY WGQGTQVTVSS (SEQ ID NO: 79) G12VQLQASGGGFVQPGGSLRLSCA ASGRTSGWYAMGWFRQAPGKER EFVSAISSRASSQEYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTATYYCAVWMKMGIEIYWGQ GTQVTVSS (SEQ ID NO: 80)NB61 VQLQASGGGFVQPGGSLRLSCA ASGTTWFNEVMGWFRQAPGKEREFVSAISGSTSWAEYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCARMSFMRAGRTPMTPMYWGQGTQVTVSS (SEQ ID NO: 93) 212B VQLQASGGGFVQPGGSLRLSCAASGDTWWSSAMGWFRQAPGKER EFVSAISFYPTEYTYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTATYYCAWIAWGPWMRTSWY WGQGTQVTVSS (SEQ ID NO: 94) 111B VQLQASGGGFVQPGGSLRLSCA ASGGTSKQYGMGWFRQAPGKER EFVSAISRQEGETIYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTATYYCAYRHVWPYPEYWGQ GTQVTVSS  (SEQ ID NO: 95)404F  VQLQASGGGFVQPGGSLRLSCA ASGRTSKNYAMGWFRQAPGKEREFVSAISWTTNQDTYYADSVKG RFTISRDNSKNTVYLQMNSLRA EDTATYYCAIWDKREISIYWGQGTQVTVSS  (SEQ ID NO: 96)

A first object of the present invention relates to a single domainantibody wherein the amino acid sequences of CDR1-IMGT, CDR2-IMGT andCDR3-IMGT have at least 90% of identity with the amino acid sequences ofthe CDR1-IMGT, CDR2-IMGT and CDR3-IMGT of the H12, 4P75, 4SP1, 4SNP36,4SNP61, 5SP10, 5SP11, 5SP58, 5SNP47, 5SNP48, 5SNP65, B6, B20, B15, B5,B71, E3, A6, G12, NB61, 212B, 111B or 404F (hs2dAb) single domainantibody.

According to the invention a first amino acid sequence having at least90% of identity with a second amino acid sequence means that the firstsequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identitywith the second amino acid sequence. Amino acid sequence identity istypically determined using a suitable sequence alignment algorithm anddefault parameters, such as BLAST P (Karlin and Altschul, 1990).

In some embodiments the single domain antibody of the present inventioncomprises the CDR1-IMGT, CDR2-IMGT and CDR3-IMGT of the H12, 4P75, 4SP1,4SNP36, 4SNP61, 5SP10, 5SP11, 5SP58, 5SNP47, 5SNP48, 5SNP65, B6, B20,B15, B5, B71, E3, A6, G12, NB61, 212B, 111B or 404F (hs2dAb) singledomain antibody.

In some embodiments, the single domain antibody of the present inventioncomprises a framework region FR1 having at least 90% of identity withSEQ ID NO:1.

In some embodiments, the single domain antibody of the present inventioncomprises a framework region FR2 having at least 90% of identity withSEQ ID NO:2.

In some embodiments, the single domain antibody of the present inventioncomprises a framework region FR3 having at least 90% of identity withSEQ ID NO:3.

In some embodiments, the single domain antibody of the present inventioncomprises a framework region FR4 having at least 90% of identity withSEQ ID NO:4.

In some embodiments the single domain antibody of the present inventioncomprises the framework regions FR1-FR4 having at least 90% of identitywith SEQ ID NO:1-4 respectively.

In some embodiments, the single domain antibody of the present inventioncomprises an amino acid sequence having at least 70% of identity withthe amino acid sequence represented by SEQ ID NO:62-80 and SEQ ID NO:93-96.

According to the invention a first amino acid sequence having at least70% of identity with a second amino acid sequence means that the firstsequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84;85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% ofidentity with the second amino acid sequence.

In some embodiments, the single domain antibody of the present inventioncomprises an amino acid sequence having at least 90% of identity withthe amino acid sequence represented by SEQ ID NO:62-80 and SEQ ID NO:93-96.

In some embodiment, the single domain antibody of the present inventioncomprises an amino acid sequence selected from the group consisting ofthe amino acid sequences represented by SEQ ID NO:62-80 and SEQ ID NO:93-96.

In some embodiment, the single domain antibody of the present inventionis fused to a heterologous polypeptide to form fusion protein. As usedherein, a “fusion protein” comprises all or part (typically biologicallyactive) of a single domain antibody of the present invention operablylinked to a heterologous polypeptide (i.e., a polypeptide other than thesame single domain antibody). Within the fusion protein, the term“operably linked” is intended to indicate that the polypeptide of theinvention and the heterologous polypeptide are fused in-frame to eachother. The heterologous polypeptide can be fused to the N-terminus orC-terminus of the single domain antibody of the invention. In someembodiment, the heterologous polypeptide is fused to the C-terminal endof the single domain antibody of the present invention.

In some embodiments, the single domain antibody of the present inventionand the heterologous polypeptide are fused to each other directly (i.e.without use of a linker) or via a linker. The linker is typically alinker peptide and will, according to the invention, be selected so asto allow binding of the single domain antibody to the heterologouspolypeptide. Suitable linkers will be clear to the skilled person basedon the disclosure herein, optionally after some limited degree ofroutine experimentation. Suitable linkers are described herein andmay—for example and without limitation—comprise an amino acid sequence,which amino acid sequence preferably has a length of 2 or more aminoacids. Typically, the linker has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acids. However, the upper limit is not critical but is chosen forreasons of convenience regarding e.g. biopharmaceutical production ofsuch fusion proteins. The linker sequence may be a naturally occurringsequence or a non-naturally occurring sequence. If used fortherapeutical purposes, the linker is preferably non-immunogenic in thesubject to which the fusion protein of the present invention isadministered. One useful group of linker sequences are linkers derivedfrom the hinge region of heavy chain antibodies as described in WO96/34103 and WO 94/04678. Other examples are poly-alanine linkersequences such as Ala-Ala-Ala. Further preferred examples of linkersequences are Gly/Ser linkers of different length including (gly4ser)3,(gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3 ser2)3.

In some embodiments, the single domain antibody of the invention isfused to an immunoglobulin domain. For example the fusion protein of thepresent invention may comprise a single domain antibody of the inventionthat is fused to an Fc portion (such as a human Fc). Said Fc portion maybe useful for increasing the half-life and even the production of thesingle domain antibody of the invention. For example the Fc portion canbind to serum proteins and thus increases the half-life on the singledomain antibody. In some embodiments, the at least one single domainantibody may also be fused to one or more (typically human) CH1, and/orCH2 and/or CH3 domains, optionally via a linker sequence. For instance,a single domain antibody fused to a suitable CH1 domain could forexample be used—together with suitable light chains—to generate antibodyfragments/structures analogous to conventional Fab fragments or F(ab′)2fragments, but in which one or (in case of an F(ab′)2 fragment) one orboth of the conventional VH domains have been replaced by a singledomain antibody of the invention. In some embodiments, one or moresingle domain antibodies of the invention may be fused to one or moreconstant domains (for example, 2 or 3 constant domains that can be usedas part of/to form an Fc portion), to an Fc portion and/or to one ormore antibody parts, fragments or domains that confer one or moreeffector functions and/or may confer the ability to bind to one or moreFc receptors. For example, for this purpose, and without being limitedthereto, the one or more further amino acid sequences may comprise oneor more CH2 and/or CH3 domains of an antibody, such as from a heavychain antibody and more typically from a conventional human chainantibody; and/or may form and Fc region, for example from IgG (e.g. fromIgG1, IgG2, IgG3 or IgG4), from IgE or from another human Ig such asIgA, IgD or IgM. For example, WO 94/04678 describes heavy chainantibodies comprising a Camelid VHH domain or a humanized derivativethereof (i.e. a single domain antibody), in which the Camelidae CH2and/or CH3 domain have been replaced by human CH2 and CH3 domains, so asto provide an immunoglobulin that consists of 2 heavy chains eachcomprising a single domain antibody and human CH2 and CH3 domains (butno CHI domain), which immunoglobulin has the effector function providedby the CH2 and CH3 domains and which immunoglobulin can function withoutthe presence of any light chains.

In some embodiments, the heterologous polypeptide is a single domainantibody. Accordingly, in some embodiments, the fusion protein of thepresent invention is a biparatopic polypeptide. As used herein, the term“biparatopic” polypeptide means a polypeptide comprising a first singledomain antibody and a second single domain antibody as herein defined,wherein these two single domain antibodies are capable of binding to twodifferent epitopes of one antigen (i.e. Rho GTPase). The biparatopicpolypeptides according to the invention are composed of single domainantibodies which have different epitope specificities, and do notcontain mutually complementary variable domain pairs which bind to thesame epitope. They do therefore not compete with each other for bindingto Rho GTPase.

In some embodiments, the heterologous polypeptide is a carrierpolypeptide. Suitable carriers are well known in the art, and include,e.g., thyroglobulin, albumins such as human serum albumin, tetanustoxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemagglutinin, influenza virus nucleoprotein; hepatitisB virus core protein, hepatitis B virus surface antigen; purifiedprotein derivative (PPD) of tuberculin from Mycobacterium tuberculosis;inactivated Pseudomonas aeruginosa exotoxin A (toxin A); Keyhole LimpetHemocyanin (KLH); filamentous hemagglutinin (FHA) of Bordetellapertussis; T helper cell (Th) epitopes of tetanus toxoid (TT) andBacillus Calmette-Guerin (BCG) cell wall; recombinant 10 kDa, 19 kDa and30-32 kDa proteins from M. leprae or from M. tuberculosis, or anycombination of these proteins; and the like.

In some embodiments, the heterologous polypeptide is a fluorescentpolypeptide. Suitable fluorescent polypeptides include, but are notlimited to, a green fluorescent protein (GFP), including, but notlimited to, a “humanized” version of a GFP, e.g., wherein codons of thenaturally-occurring nucleotide sequence are changed to more closelymatch human codon bias; a GFP derived from Aequoria victoria or aderivative thereof, e.g., a “humanized” derivative such as Enhanced GFP,which are available commercially, e.g., from Clontech, Inc.; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle etal. (2001) J. Protein Chem. 20:507-519; “humanized” recombinant GFP(hrGFP) (Stratagene); any of a variety of fluorescent and coloredproteins from Anthozoan species, as described in, e.g., Matz et al.(1999) Nature Biotechnol. 17:969-973; and the like.

In some embodiments, the heterologous polypeptide is an enzyme.Typically, said enzyme may be selected from the group consisting ofβ-galactosidase, alkaline phosphatase, luciferase, and horse radishperoxidise). Where the heterologous polypeptide is an enzyme that yieldsa detectable product, the product can be detected using an appropriatemeans, e.g., β-galactosidase can, depending on the substrate, yieldcolored product, which is detected spectrophotometrically, or afluorescent product; luciferase can yield a luminescent productdetectable with a luminometer; etc.

In some embodiments, the heterologous polypeptide is a polypeptide thatfacilitates purification or isolation of the fusion protein, e.g., metalion binding polypeptides such as 6H is tags (e.g., acetylated Tat/6His),or glutathione-S-transferase.

In some embodiments, the heterologous polypeptide is a cell-penetratingpeptide. The term “cell-penetrating peptides” is well known in the artand refers to cell permeable sequences or membranous penetratingsequences such as penetratin, TAT mitochondrial penetrating sequence andcompounds described in Bechara and Sagan, 2013; Jones and Sayers, 2012;Khafagy el and Morishita, 2012; and Malhi and Murthy, 2012. In aparticular embodiment, the heterologous polypeptide is theTransactivator of Transcription (TAT) cell penetrating sequenceoriginally derived from the cell-penetrating HIV tat peptide.

In some embodiments, the heterologous polypeptide is a domain of anubiquitin ligase, such as E3 ubiquitin ligase. Examples of various E3ubiquitin ligase domains include RING, HECT, U-box, RIBRR, F-box domain,DCAF domain, DDS2, HIF-mimetic peptides, IkB-mimetic sequences, BTBdomain, or combination thereof. These E3 ligase domains facilitateubiquitination, and when fused with the single domain antibody of thepresent allows for the degradation of the antigen-antibody complex. AnyE3 ligase domains including E2 binding domains known or later discoveredor developed can be used. Recombinant E3 ligase domains can be used. Insome embodiment, the heterologous polypeptide is a F-box domain. TheF-box domain is typically a protein motif of approximately 50 aminoacids. The F-box domain tethers the F-box protein to other components ofthe SCF complex by binding the core SCF component, Skp1.

In some embodiments, the heterologous polypeptide is a switchabledomain, which can be activated by a small molecule or byphotoactivation. Examples of small molecule switchable system includehormone ligand binding domain such as ERalpha LBD, Auxin AID system,HaloTag2 derivative system HyT or HALTS, FKB-FRB rapamycin or shield1systems. Examples of photoactivation systems include Lov2 domain,PhyB-PIF, Cry2, UVR8, or Dronpa. These switchable systems are typicallyused for a precise spatial or temporal control of protein functions byconformational changed or relocalisation.

The single domain antibody of the present invention (fused or not to theheterologous polypeptide) is produced by any technique known in the art,such as, without limitation, any chemical, biological, genetic orenzymatic technique, either alone or in combination. For example,knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said single domain antibody (fused or not tothe heterologous polypeptide), by standard techniques for production ofpolypeptides. For instance, they can be synthesized using well-knownsolid phase method, preferably using a commercially available peptidesynthesis apparatus (such as that made by Applied Biosystems, FosterCity, Calif.) and following the manufacturer's instructions.Alternatively, the single domain antibody of the present invention(fused or not to the heterologous polypeptide) can be synthesized byrecombinant DNA techniques well-known in the art. For example, thesingle domain of the present invention (fused or not to the heterologouspolypeptide) can be obtained as DNA expression products afterincorporation of DNA sequences encoding the single domain antibody(fused or not to the heterologous polypeptide) into expression vectorsand introduction of such vectors into suitable eukaryotic or prokaryotichosts that will express the desired single domain antibody, from whichthey can be later isolated using well-known techniques. A variety ofexpression vector/host systems may be utilized to contain and expressthe single domain antibody of the present invention (fused or not to theheterologous polypeptide). These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors (Giga-Hama et al., 1999);insect cell systems infected with virus expression vectors (e.g.,baculovirus, see Ghosh et al., 2002); plant cell systems transfectedwith virus expression vectors (e.g., cauliflower mosaic virus, CaMV;tobacco mosaic virus, TMV) or transformed with bacterial expressionvectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); oranimal cell systems. Those of skill in the art are aware of varioustechniques for optimizing mammalian expression of proteins, see e.g.,Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful inrecombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of thepeptide substrates or fusion polypeptides in bacteria, yeast and otherinvertebrates are known to those of skill in the art and a brieflydescribed herein below. Mammalian host systems for the expression ofrecombinant proteins also are well known to those of skill in the art.Host cell strains may be chosen for a particular ability to process theexpressed protein or produce certain post-translation modifications thatwill be useful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like have specific cellular machinery and characteristic mechanismsfor such post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.In the recombinant production of the single domain antibody of thepresent invention (fused or not to the heterologous polypeptide), itwould be necessary to employ vectors comprising polynucleotide moleculesfor encoding said single domain antibody. Methods of preparing suchvectors as well as producing host cells transformed with such vectorsare well known to those skilled in the art. The polynucleotide moleculesused in such an endeavour may be joined to a vector, which generallyincludes a selectable marker and an origin of replication, forpropagation in a host. These elements of the expression constructs arewell known to those of skill in the art. Generally, the expressionvectors include DNA encoding the given protein being operably linked tosuitable transcriptional or translational regulatory sequences, such asthose derived from a mammalian, microbial, viral, or insect genes.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, mRNA ribosomal binding sites, and appropriatesequences which control transcription and translation. The terms“expression vector,” “expression construct” or “expression cassette” areused interchangeably throughout this specification and are meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The choice of a suitableexpression vector for expression of single domain antibody of thepresent invention will of course depend upon the specific host cell tobe used, and is within the skill of the ordinary artisan. Expressionrequires that appropriate signals be provided in the vectors, such asenhancers/promoters from both viral and mammalian sources that may beused to drive expression of the nucleic acids of interest in host cells.Usually, the nucleic acid being expressed is under transcriptionalcontrol of a promoter. Typically, the nucleotide sequences are operablylinked when the regulatory sequence functionally relates to the DNAencoding the protein of interest (e.g., a single domain antibody). Thus,a promoter nucleotide sequence is operably linked to a given DNAsequence if the promoter nucleotide sequence directs the transcriptionof the sequence. They may then, if necessary, be purified byconventional procedures, known in themselves to those skilled in theart, for example by fractional precipitation, in particular ammoniumsulphate precipitation, electrophoresis, gel filtration, affinitychromatography, etc. In particular, conventional methods for preparingand purifying recombinant proteins may be used for producing theproteins in accordance with the invention.

A further object of the present invention relates to a nucleic acidmolecule which encodes for a single domain antibody of the presentinvention (fused or not to the heterologous polypeptide).

As used herein, the term “nucleic acid molecule” has its general meaningin the art and refers to a DNA or RNA molecule. However, the termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the nucleic acid molecule of the present inventionis included in a suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or a viral vector. So, a further object ofthe invention relates to a vector comprising a nucleic acid encoding fora single domain antibody of the invention (fused or not to theheterologous polypeptide). Typically, the vector is a viral vector whichis an adeno-associated virus (AAV), a retrovirus, bovine papillomavirus, an adenovirus vector, a lentiviral vector, a vaccinia virus, apolyoma virus, or an infective virus. In some embodiments, the vector isan AAV vector. As used herein, the term “AAV vector” means a vectorderived from an adeno-associated virus serotype, including withoutlimitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, andmutated forms thereof. AAV vectors can have one or more of the AAVwild-type genes deleted in whole or part, preferably the rep and/or capgenes, but retain functional flanking ITR sequences. Retroviruses may bechosen as gene delivery vectors due to their ability to integrate theirgenes into the host genome, transferring a large amount of foreigngenetic material, infecting a broad spectrum of species and cell typesand for being packaged in special cell-lines. In order to construct aretroviral vector, a nucleic acid encoding a gene of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line is constructed containing the gag, pol,and/or env genes but without the LTR and/or packaging components. When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media. The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. Lentiviruses are complexretroviruses, which, in addition to the common retroviral genes gag,pol, and env, contain other genes with regulatory or structuralfunction. The higher complexity enables the virus to modulate its lifecycle, as in the course of latent infection. Some examples of lentivirusinclude the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the SimianImmunodeficiency Virus (SIV). Lentiviral vectors have been generated bymultiply attenuating the HIV virulence genes, for example, the genesenv, vif, vpr, vpu and nef are deleted making the vector biologicallysafe. Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos.6,013,516 and 5,994,136, both of which are incorporated herein byreference. In general, the vectors are plasmid-based or virus-based, andare configured to carry the essential sequences for incorporatingforeign nucleic acid, for selection and for transfer of the nucleic acidinto a host cell. The gag, pol and env genes of the vectors of interestalso are known in the art. Thus, the relevant genes are cloned into theselected vector and then used to transform the target cell of interest.Recombinant lentivirus capable of infecting a non-dividing cell whereina suitable host cell is transfected with two or more vectors carryingthe packaging functions, namely gag, pol and env, as well as rev and tatis described in U.S. Pat. No. 5,994,136, incorporated herein byreference. This describes a first vector that can provide a nucleic acidencoding a viral gag and a pol gene and another vector that can providea nucleic acid encoding a viral env to produce a packaging cell.Introducing a vector providing a heterologous gene into that packagingcell yields a producer cell which releases infectious viral particlescarrying the foreign gene of interest. The env preferably is anamphotropic envelope protein which allows transduction of cells of humanand other species. Typically, the nucleic acid molecule or the vector ofthe present invention include “control sequences”, which referscollectively to promoter sequences, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, internal ribosome entry sites (“IRES”),enhancers, and the like, which collectively provide for the replication,transcription and translation of a coding sequence in a recipient cell.Not all of these control sequences need always be present so long as theselected coding sequence is capable of being replicated, transcribed andtranslated in an appropriate host cell. Another nucleic acid sequence,is a “promoter” sequence, which is used herein in its ordinary sense torefer to a nucleotide region comprising a DNA regulatory sequence,wherein the regulatory sequence is derived from a gene which is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. Transcription promoters can include“inducible promoters” (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), “repressible promoters” (where expression ofa polynucleotide sequence operably linked to the promoter is induced byan analyte, cofactor, regulatory protein, etc.), and “constitutivepromoters”.

A further object of the present invention relates to a host celltransformed with the nucleic acid molecule of the present invention. Theterm “transformation” means the introduction of a “foreign” (i.e.extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. A host cell that receives and expressesintroduced DNA or RNA has been “transformed”. For instance, as disclosedabove, for expressing and producing the single domain antibody of thepresent invention, prokaryotic cells and, in particular E. coli cells,will be chosen. Actually, according to the invention, it is notmandatory to produce the single domain antibodys of the presentinvention in a eukaryotic context that will favour post-translationalmodifications (e.g. glycosylation). Typically, the host cell may besuitable for producing the single domain antibody of the presentinvention (fused or not to the heterologous polypeptide) as describedabove. In some cases, the host cell is used as a research tool, to studye.g. the impact of the Rho GTPase activation or inactivation (e.g.functional knockdown) in a cell of interest as described in the EXAMPLE.In some embodiments, the host cells is isolated from a mammalian subjectwho is selected from a group consisting of: a human, a horse, a dog, acat, a mouse, a rat, a cow and a sheep. In some embodiments, the hostcell is a human cell. In some embodiments, the host cell is a cell inculture. The cells may be obtained directly from a mammal (preferablyhuman), or from a commercial source, or from tissue, or in the form forinstance of cultured cells, prepared on site or purchased from acommercial cell source and the like. The cells may come from any organincluding but not limited to the blood or lymph system, from muscles,any organ, gland, the skin, brain, lung . . . In some embodiments, thecells are selected from the group consisting of epithelial cells, neuralcells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes,chondrocytes, hepatocytes, B-cells, T-cells, erythrocytes, macrophages,monocytes, fibroblasts, muscle cells, vascular smooth muscle cells,hepatocytes, splenocytes, pancreatic β cells . . . . In someembodiments, the host cell is a cancer cell. Typically, the cancer cellsare isolated from a cancer selected from the group consisting of breastcancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, coloncancer, melanoma, malignant melanoma, ovarian cancer, brain cancer,primary brain carcinoma, head-neck cancer, glioma, glioblastoma, livercancer, bladder cancer, non-small cell lung cancer, head or neckcarcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma,small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicularcarcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma,colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, chronic granulocytic leukemia, acute granulocyticleukemia, acute myelogenous leukemia, chronic myelogenous leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, and retinoblastoma. In some embodiment, the hostcells is a stem cell. As used herein, the term “stem cell” refers to anundifferentiated cell that can be induced to proliferate. The stem cellis capable of self-maintenance or self-renewal, meaning that with eachcell division, one daughter cell will also be a stem cell. Stem cellscan be obtained from embryonic, post-natal, juvenile, or adult tissue.Stem cells can be pluripotent or multipotent. The term “progenitorcell,” as used herein, refers to an undifferentiated cell derived from astem cell, and is not itself a stem cell. Some progenitor cells canproduce progeny that are capable of differentiating into more than onecell type. Stem cells include pluripotent stem cells, which can formcells of any of the body's tissue lineages: mesoderm, endoderm andectoderm. Therefore, for example, stem cells can be selected from ahuman embryonic stem (ES) cell; a human inner cell mass (ICM)/epiblastcell; a human primitive ectoderm cell, a human primitive endoderm cell;a human primitive mesoderm cell; and a human primordial germ (EG) cell.Stem cells also include multipotent stem cells, which can form multiplecell lineages that constitute an entire tissue or tissues, such as butnot limited to hematopoietic stem cells or neural precursor cells. Stemcells also include totipotent stem cells, which can form an entireorganism. In some embodiment, the stem cell is a mesenchymal stem cell.The term “mesenchymal stem cell” or “MSC” is used interchangeably foradult cells which are not terminally differentiated, which can divide toyield cells that are either stem cells, or which, irreversiblydifferentiate to give rise to cells of a mesenchymal cell lineage, e.g.,adipose, osseous, cartilaginous, elastic and fibrous connective tissues,myoblasts) as well as to tissues other than those originating in theembryonic mesoderm (e.g., neural cells) depending upon variousinfluences from bioactive factors such as cytokines. In someembodiments, the stem cell is a partially differentiated ordifferentiating cell. In some embodiments, the stem cell is an inducedpluripotent stem cell (iPSC), which has been reprogrammed orde-differentiated. Stem cells can be obtained from embryonic, fetal oradult tissues.

The single domain antibody of the present invention (fused or not to theheterologous polypeptide) may be used in the research and diagnosticfield. For instance, the single domain antibody of the invention is thusparticularly suitable for detecting the present of an activated form ofa Rho GTPase, said detection may find usefulness for research ordiagnostic purpose.

Thus, a further aspect of the present invention, there is provided amethod of detecting the present of a least one activated form of a RhoGTPase (e.g. RhoA, RhoB, and/or RhoC) comprising the steps of i) a)obtaining a sample from a subject, ii) contacting, in vitro, the samplewith a single domain antibody of the present invention (fused or not tothe heterologous polypeptide), iii) detecting the binding of said singledomain antibody to said sample, and iv) comparing the binding detectedin step (iii) with a standard, wherein a difference in binding relativeto said sample indicated the presence of the activated form of the RhoGTPase. Typically, the detection is performed with any suitable meanssuch as a microscope or an automated analysis system.

As used herein the term “sample” encompasses a variety of sample typesobtained from a subject and can be used in a diagnostic or researchassay. Biological samples include but are not limited to blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom, and theprogeny thereof. In some embodiments, the sample is a tumor tissuesample. The term “tumor sample” means any tissue sample derived from thetumor of the subject. The tissue sample is obtained for the purpose ofthe in vitro evaluation and typically results from biopsy performed in atumor of the subject. The sample can be fresh, frozen, or embedded(e.g., FFPE biopsy).

Accordingly, in some embodiments, the single domain antibody of thepresent invention (fused or not to the heterologous polypeptide) isconjugated with a detectable label. Suitable detectable labels include,for example, a radioisotope, a fluorescent label, a chemiluminescentlabel, an enzyme label, a bio luminescent label or colloidal gold.Methods of making and detecting such detectably-labeled immunoconjugatesare well-known to those of ordinary skill in the art, and are describedin more detail below. For instance, the detectable label can be aradioisotope that is detected by autoradiography. Isotopes that areparticularly useful for the purpose of the present invention are 3H,125I, 131I, 35S and 14C. The single domain antibody of the presentinvention (fused or not to the heterologous polypeptide) can also belabeled with a fluorescent compound. The presence of afluorescently-labeled single domain antibody of the present invention isdetermined by exposing the immuno conjugate to light of the properwavelength and detecting the resultant fluorescence. Fluorescentlabeling compounds include fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine and Alexa Fluor dyes. Alternatively, the single domainantibody of the present invention can be detectably labeled by couplingsaid single domain antibody to a chemiluminescent compound. The presenceof the chemiluminescent-tagged immuno conjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester. Similarly, a bio luminescentcompound can be used to label the single domain antibody of the presentinvention. Bioluminescence is a type of chemiluminescence found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. The presence of a bioluminescentprotein is determined by detecting the presence of luminescence.Bioluminescent compounds that are useful for labeling include luciferin,luciferase and aequorin. Typically, when the single domain antibody isfused to a fluorescent polypeptide as described above, the presence ofthe fusion protein can be detected with any means well known in the artsuch as a microscope or microscope or automated analysis system.Typically, when the single domain antibody is fused to an enzyme then,the fusion protein is incubated in the presence of the appropriatesubstrate, the enzyme moiety reacts with the substrate to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase. Those of skill in the art will know of other suitablelabels which can be employed in accordance with the present invention.The binding of marker moieties to anti-the single domain antibody of thepresent invention is accomplished using standard techniques known to theart. Typical methodology in this regard is described by Kennedy et al.,Clin. Chim. Acta 70: 1, 1976; Schurs et al., Clin. Chim. Acta 81: 1,1977; Shih et al., Int′U. Cancer 46: 1101, 1990; Stein et al, CancerRes. 50: 1330, 1990; and Coligan, supra. Moreover, the convenience andversatility of immunochemical detection can be enhanced by using singledomain antibodies of the present invention (fused or not to theheterologous polypeptide) that have been conjugated with avidin,streptavidin, and biotin. {See, e.g., Wilchek et al. (eds.),“Avidin-Biotin Technology”, Methods In Enzymology (Vol. 184) (AcademicPress 1990); Bayer et al., “Immunochemical Applications of Avidin-BiotinTechnology,” in Methods In Molecular Biology (Vol. 10) 149-162 (Manson,ed., The Humana Press, Inc. 1992).) In some embodiments, the presence ofthe single domain antibody (fused or not to the heterologouspolypeptide) is detected with a secondary antibody that is specific forthe single antibody of the present invention (fused or not to theheterologous polypeptide). Typically said secondary is labeled by samemethods as described above. For instance when the single domain antibodyof the present invention is fused to a tag (e.g. histidine tag) thesecondary antibody is specific for said tag. Methods for performingimmunoassays are well-established. {See, e.g., Cook and Self,“Monoclonal Antibodies in Diagnostic Immunoassays”, in MonoclonalAntibodies: Production, Engineering, and Clinical Application 180-208(Ritter and Ladyman, eds., Cambridge University Press 1995); Perry, “TheRole of Monoclonal Antibodies in the Advancement of ImmunoassayTechnology”, in Monoclonal Antibodies: Principles and Applications107-120 (Birch and Lennox, eds., Wiley-Liss, Inc. 1995); Diamandis,Immunoassay (Academic Press, Inc. 1996)).

The single domain antibody of the present invention (fused or not to theheterologous polypeptide) and nucleic acid molecules encoding thereofcan be used as medicament. In particular, the nucleic acid molecules ofthe present invention (inserted or not into a vector) are particularlysuitable for gene therapy.

In some embodiments, the single domain antibody and nucleic acidmolecules of the present invention (inserted or not into a vector) areparticularly suitable for the treatment of cancer. As used herein, theterm “cancer” has its general meaning in the art and includes, but isnot limited to, solid tumors and blood borne tumors. The term cancerincludes diseases of the skin, tissues, organs, bone, cartilage, bloodand vessels. The term “cancer” further encompasses both primary andmetastatic cancers. Examples of cancers that may treated by methods andcompositions of the invention include, but are not limited to, cancercells from the bladder, blood, bone, bone marrow, brain, breast, colon,oesophagus, gastrointestine, gum, head, kidney, liver, lung,nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, oruterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous; adenocarcinoma; mucoepidermoid carcinoma;cystadenocarcinoma; papillary cystadenocarcinoma; papillary serouscystadenocarcinoma; mucinous cystadenocarcinoma; mucinousadenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma;medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget'sdisease, mammary; acinar cell carcinoma; adenosquamous carcinoma;adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarianstromal tumor, malignant; thecoma, malignant; granulosa cell tumor,malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydigcell tumor, malignant; lipid cell tumor, malignant; paraganglioma,malignant; extra-mammary paraganglioma, malignant; pheochromocytoma;glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficialspreading melanoma; malig melanoma in giant pigmented nevus; epithelioidcell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibroushistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma;rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma;stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor;nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant;brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma;mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma,malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma,malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi'ssarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

Accordingly a further object of the present invention relates to amethod for treating cancer in a subject in need thereof comprisingadministering the subject with a therapeutically effective amount of asingle domain antibody of the present invention (fused or not to theheterologous polypeptide) or a nucleic acid molecule of the presentinvention which is inserted or not in to a vector as above described.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By a “therapeutically effective amount” is meant asufficient amount of the single domain antibody or the nucleic acidmolecule of the present invention thereof for the treatment of thedisease at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood that the total daily usage of theactive agent will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective doselevel for any particular subject will depend upon a variety of factorsincluding the age, body weight, general health, sex and diet of thesubject; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificpolypeptide employed; and like factors well known in the medical arts.For example, it is well known within the skill of the art to start dosesof the compound at levels lower than those required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved. However, the daily dosage of theproducts may be varied over a wide range from 0.01 to 1,000 mg per adultper day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0,2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the activeingredient for the symptomatic adjustment of the dosage to the subjectto be treated. A medicament typically contains from about 0.01 mg toabout 500 mg of the active ingredient, preferably from 1 mg to about 100mg of the active ingredient. An effective amount of the drug isordinarily supplied at a dosage level from 0.0002 mg/kg to about 20mg/kg of body weight per day, especially from about 0.001 mg/kg to 7mg/kg of body weight per day.

According to the invention, the single domain antibody (fused or not tothe heterologous polypeptide) or the nucleic acid molecule (inserted ornot into a vector) of the present invention is administered to thesubject in the form of a pharmaceutical composition. Typically, thesingle domain antibody or the nucleic acid molecule (inserted or notinto a vector) of the present invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form pharmaceuticalcompositions. “Pharmaceutically” or “pharmaceutically acceptable” referto molecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to a mammal,especially a human, as appropriate. A pharmaceutically acceptablecarrier or excipient refers to a non-toxic solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. In the pharmaceutical compositions of the present invention fororal, sublingual, subcutaneous, intramuscular, intravenous, transdermal,local or rectal administration, the active principle, alone or incombination with another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms. Typically, the pharmaceutical compositions containvehicles which are pharmaceutically acceptable for a formulation capableof being injected. These may be in particular isotonic, sterile, salinesolutions (monosodium or disodium phosphate, sodium, potassium, calciumor magnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions. The pharmaceutical forms suitablefor injectable use include sterile aqueous solutions or dispersions;formulations including sesame oil, peanut oil or aqueous propyleneglycol; and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions comprisingcompounds of the invention as free base or pharmacologically acceptablesalts can be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The single domainantibody or the nucleic acid molecule (inserted or not into a vector) ofthe present invention can be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. The carrier can also be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin. Sterile injectable solutions are prepared byincorporating the active compounds in the required amount in theappropriate solvent with several of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the typical methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparation of more, or highlyconcentrated solutions for direct injection is also contemplated, wherethe use of DMSO as solvent is envisioned to result in extremely rapidpenetration, delivering high concentrations of the active agents to asmall tumor area. Upon formulation, solutions will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIGS. 1A-1C: The H12 hs2dAb is selective for Rho in its activeconformation. (A) H12 is a conformational hs2dAb binding only to the GTPbound, activated state of RhoA GTPase. ELISA revealing recombinantGST-RhoA wild type loaded with either 100 μM GTP gamma S (Black)) or 1mM GDP (White), or a purified GST fusion of a RhoA constitutively activemutant Q63L (Check). (B) A CBD tagged H12 pull down from HeLa cellextract loaded with 100 μM GTP gamma S (GTP) or with 1 mM GDP as inputs.Western blot reveals RhoA at similar level in 5% of both input but onlyon the GTP loaded extract in the CBD-H12 pull down. D5 anti tubulin wasa used as negative control and the standard GST-RBD (Rho binding domainof Rhotekin) as a positive control of active Rho pull down. (C)immunofluorescence on HeLa cells overexpressing GFP-RhoAT19N inactivemutant or GFPRhoAQ63L. H12 staining detected using a myc tag antibodyrevealed only cells overexpressing the constitutively active mutant witha pattern similar to the GFP fluorescence.

FIG. 2: The H12 hs2dAb is able to perturb endogenous Rho activity whenexpressed in the cytosol. HeLa cells transfected with a control nonrelevant hs2dAb or the clone H12 anti Rho-GTP expressed as GFP fusion.Cells were fixed 20 h hours post transfection and stained with DAPI andAlexa 594 phalloidin to label actin stress fibers.

FIGS. 3A-3E: Characterization of selected F-Ib. (A) mCherry fluorescencequantification by flow cytometry in Hm and HmB cell lines. After 48 h ofF-Ib transfection in the Hm and the HmB cell lines, the mCherryfluorescence was quantified in the transfected subpopulation and in thenon transfected subpopulation for each F-Ib. The ratio of each median offluorescence (transfected versus non transfected population) gives apercentage of mCherry fluorescence intensity for one F-Ib. (B) Fboxdomain is responsible of RhoB degradation in HmB cell line. F-hs2dAb andhs2dAb were transfected in HmB cell line. The median of mCherryfluorescence was determined by flow cytometry as in (A). A decrease ofmCherry fluorescence is only observed with the F-Ib whereas the hs2dAbalone are not able to induce such decay. (C) Degradation in HmB cellline is proteasome dependent. HmB cells were transfected with F-Ib andtreated 36 h with 1 μM of MG132 (a proteasome inhibitor) or DMSO. MG132treatment restores the fluorescence level nearly to the control level.Medians of fluorescence are normalized to the NR control. (D) F-H12 andF-B5 degrade Rac1 mutant. After 48 h of F-Ib transfection, the mCherryfluorescence was quantified by flow cytometry in Hm, HmB andH2B-mCherry-Rac1L63 cell lines as describe in (A) and median offluorescence for each cell line was normalized to the NR control. F-H12and F-B5 induce a significant decrease of mCherry fluorescence inRac1L63 cell line compared to the other F-Ib. (E) All RhoB positive F-Ibare conformational sensitive, selective towards the active mutantRhoBL63. As describe above, the mCherry fluorescence was quantified byflow cytometry after F-Ib transfection in Hm, HmB andH2B-mCherry-RhoBN19 cell lines and median of fluorescence for each cellline was normalized to the NR control. No significant decrease inmCherry fluorescence was observed in RhoBN19 cell line compared to thecontrol cell line Hm for each F-Ib.

FIGS. 4A-4E: Endogenous RhoB cellular activation knockdown. (A) Hela S3cells were transfected 48 h with F-Ib plasmids. A GST-RBD pulldown wasperformed for each F-Ib to control Rho-GTP level (lines RhoB-GTP,RhoA-GTP and RhoC-GTP) and the total level of Rho proteins was revealedby loading 2% of input (lines total RhoB, total RhoA and total RhoC).F-Ib production is shown with myc tag revelation and tubulin is theloading control. (B) Quantification of three independent GST-RBDpulldown experiments. F-B6 seems to degrade more selectively RhoB-GTPthan RhoA or RhoC. F-H12 and F-B15 are pan Rho binders. Relativeactivity was calculated as the ratio between GTP level to input levelnormalized to tubulin. Normalized means±SEM are shown. (C) RhoBactivation kinetic after an EGF treatment. HeLa S3 cells weretransfected with F-NR control 48 h including 24 h of serum starvation.At 48 h of transfection, cells were treated at an EGF concentration of50 ng.mL-1 for indicated times. A GST-RBD pulldown was performed tomonitor the Rho-GTP induction following this treatment. RhoB isactivated within 5 min until 30 min and a second wave of activation isshown at 120 min. RhoA and RhoC are activated only between 5 and 30 minwith a maximum at 5 min. (C, D, E) After 15 min of EGF treatment and 48h of cells transfection by F-Ib, RhoB-GTP (C), RhoA-GTP (D) and RhoC-GTP(E) levels were checked. F-H12 and F-B6 are able to inhibit RhoBactivation following an EGF treatment compared to the negative control.F-H12 inhibits partially RhoA activation (50%) and nearly decreasesRhoC-GTP level to the basal level (without treatment) under EGFtreatment whereas F-B6 has no inhibitory effect after EGF treatment onthese two RhoGTPases activation. Quantification is shown with normalizedmeans±SEM.

EXAMPLES Example 1: Selection of Conformation-Sensitive Antibodies

One of the main advantages of full in vitro immunization using displaytechnologies is the control of antigen conformation and concentration inorder to drive selection towards the desired outcome. For example,selection schemes can be devised to improve the recovery of highaffinity binders endowed with low off-rate kinetics, to target specificepitopes, or to identify conformation sensitive-binders. Recombinantantibody fragment library screening have for example provided severalbinders targeting selectively the active conformation of small GTPase.We hypothesized that our synthetic library (described inPCT/EP2014/073713) had enough diversity and functionality to enable theidentification of selective conformational binders. We carried outsubtractive panning to select conformation-specific antibodies directedagainst small GTPases from the Rho subfamily. Small GTPases aremolecular switch that cycle between an inactive and an active state whenbound to GDP or GTP nucleotides respectively. Mutant of small GTPasescan be designed that adopt stably an active or inactive conformation. Aconstitutively active mutant (e.g. RhoA Q63L, RhoB Q63L or RhoC Q63L)was expressed in HEK293 as bait then freshly pulled down for panning topreserve its native conformation. To enrich in phage specific forGTP-bound RhoA, a depletion step was introduced from the second round ofpanning using GDP-bound RhoA proteins, to remove generic binders beforeselecting against the active mutant. After four rounds of selection,clones were analyzed using phage ELISA against either the Rho GTPasebound to GTPγS (a non-hydrolysable analogue of GTP)-loaded Rho GTPase orGDP-loaded Rho GTPase. The basic features of the selected single domainantibodies are depicted in Table 1:

TABLE 1 basic features of the selected single domain anti bodies: Kon(10{circumflex over ( )}6 Koff (10{circumflex over ( )}−3 Name ELISA IFIP IB (2SHA) M−1 · sec−1) sec−1) Kd (nM) H12 X X X X RhoA 4.81+5 1.28−4 2.65−10 = 0.265 nM  Q63L RhoB Q63L 2.24+5 3.59−4 1.57−9 = 1.57 nM RhoCQ63L 1.12+6 5.41−5  4.79−11 = 0.0479 nM RhoA T19N négatif négatifnégatif Rac Q61L 7.53+5 2.55−4 3.3−10 = 0.33 nM 4P75 X X ND ND RhoA Q63LND ND ND RhoB Q63L ND ND ND RhoC Q63L ND ND ND RhoA T19N ND ND ND RacQ61L ND ND ND 4SP1 X X X X RhoA Q63L ND ND ND RhoB Q63L ND ND ND RhoCQ63L ND ND ND RhoA T19N ND ND ND Rac Q61L ND ND ND 4SNP36 X 0 X X RhoAQ63L 1.76+6 5.22−4 2.96−10 = 0.296 nM RhoB Q63L 2.99+6 8.34−4 2.78−10 =0.278 nM RhoC Q63L 6.52+6 5.42−4  8.31−11 = 0.0831 nM RhoA T19N ND ND NDRac Q61L ND ND ND 4SNP61 X 0 X X RhoA Q63L 1.10+6 0.0013 1.21−9 = 1.21nM RhoB Q63L 7.22+5 0.0033 4.68−9 = 4.68 nM RhoC Q63L 8.75+5 0.00465.30−9 = 5.30 nM RhoA T19N ND ND ND Rac Q61L ND ND ND 5SP10 X 0 X X RhoAQ63L ND ND ND RhoB Q63L ND ND ND RhoC Q63L ND ND ND RhoA T19N ND ND NDRac Q61L ND ND ND 5SP11 X 0 X X RhoA Q63L ND ND ND RhoB Q63L ND ND NDRhoC Q63L ND ND ND RhoA T19N ND ND ND Rac Q61L ND ND ND 5SP58 X 0 X XRhoA Q63L ND ND ND RhoB Q63L ND ND ND RhoC Q63L ND ND ND RhoA T19N ND NDND Rac Q61L ND ND ND 5SNP47 X 0 X X RhoA Q63L ND ND ND RhoB Q63L ND NDND RhoC Q63L ND ND ND RhoA T19N ND ND ND Rac Q61L ND ND ND 5SNP48 X 0 XX RhoA Q63L 8.14+5 6.86−4 8.4210 = 0.84 nM RhoB Q63L 4.62+5 0.00245.21−9 = 5.21 nM RhoC Q63L 1.72+6 9.01−4 5.24−10 = 0.524 nM RhoA T19Nnégatif négatif négatif Rac Q61L ND ND ND 5SNP65 X 0 X X RhoA Q63L8.70+5 5.22−4 6.00−10 = 0.600 nM RhoB Q63L 2.53+5 5.99−4 2.36−9 = 2.36nM RhoC Q63L 1.71+6 0.001  6.08−10 = 0.608 nM RhoA T19N négatif négatifnégatif Rac Q61L ND ND ND B6 ND X X X RhoA Q63L 1.05 0.8   1.3125 nMRhoB Q63L 1.1  1.55  0.70969 nM  RhoC Q63L 1.45 0.625   2.32 nM (ND =non determined)

Example 2: Functional Characterization of the H12 Antibody

The clone H12 was further analyzed by ELISA, using in this case thesoluble form of the antibody, on several purified Rho proteins expressedas GST fusion in E. coli. We showed that the H12 hs2dAb efficientlybound to the constitutively active mutant RhoA_(L63) as well as to wildtype RhoA loaded with GTPγS. In contrast, no binding was observed to theinactive RhoA_(N19) mutant or to GDP-loaded wild type RhoA (FIG. 1A). Wethen tested whether H12 was able to specifically pull-down GTP-loadedRhoA from mammalian cell extracts. A CBD tagged H12 construct expressedin E. coli was immobilized on chitin beads and incubated with a HeLacell extract pre-treated with either GTPγS or GDP. The Rho bindingdomain of Rhotekin fused to GST (GST-RBD) was used as a control. Thisdomain is known to bind to the active conformation of Rho GTPase and isthe standard method to assay Rho activity up to now. The H12 hs2dAb wasfound to be highly selective of Rho loaded with GTPγS, giving no signalon the GDP loaded extract (FIG. 1B). We next tested whether H12specifically detected RhoA active conformation in immunofluorescence.HeLa cells expressing the GFP-RhoA_(L63) active mutant or the inactiveGFP-RhoA, were fixed and stained with the H12 hs2dAb.

Overexpression of the inactive mutant GFP-RhoA_(N19), which has nodominant negative effect on RhoA pathway nor on cell shape, did not leadto an increased signal over the background of untransfected cells. Incontrast, a strong staining was selectively obtained on cells expressingthe GFP-RhoA_(L63) active mutant. Note that these cells display bundledactin stress fibers, a characteristic phenotype linked to enhanced RhoAactivity (FIG. 1B). Altogether these results showed that the H12 hs2dAbis selective for Rho in its active conformation.

Furthermore our results suggest that the H12 antibody was able toperturb endogenous Rho activity when expressed in the cytosol. First, weco-expressed H12-GFP in HeLa cells together with either the RhoA_(N19)inactive mutant or with the RHoA_(L63) constitutively active one andcarried out a co-immunoprecipitation experiment using an anti-GFPmonoclonal antibody. Active RhoA was co-immunoprecipated with H12-GFPwhile inactive RhoA was not. This showed that H12 worked as an intrabodyand kept its conformation sensitivity in the cytosol. Because RhoGTPases are involved in signaling pathways that promotes the actincytoskeleton polymerization we looked at functional effects induced byH12 overexpression. In contrast to untransfected cells or cellstransfected with various non-relevant GFP fused hs2dAb, we observed thatcells expressing H12-GFP were totally devoid of actin stress fibers(FIG. 2). This alteration in actin filament organization was associatedwith marked changed in cell shape characteristic of loss ofintracellular mechanical forces and tension (FIG. 2). As RhoA plays amajor role in activating myosin II and actin cytoskeletonreorganization, our results suggested that H12 efficiently perturbedRho-dependent signaling, mimicking the effects induced by the C3exoenzyme Rho inhibitor.

Example 3: Functionalization Conformational Intrabodies to Target RhoBActivity

Direct Selection of Intrabodies by Visual Screen of Fluorescent ProteinKnock Down

In the goal of interfering with RhoB activity in cells usingintrabodies, we established a strategy starting with a phage displayselection then followed by in-cell screening aiming at theidentification of a functional inhibitory intrabody. In the past decadewe established sophisticated phage display selection scheme in order toisolate binders discriminating the GTP conformation of Rho proteins. Topreserve the native conformation of RhoB during the selection, baitantigens were expressed in mammalian cells and freshly extracted andused in the nanomolar range during the incubation with the NaLi-H1library phages. A competitive panning selection was carried out using aconstitutively active mutant RhoBL63 after a preclearing step in thepresence of an excess of GDP loaded wild type RhoB to enrich in bindersmore selective towards RhoB than its closest homologs. After two roundof enrichment, we added a 5 molar excess of RhoAL63 and RhoCL63 tofurther compete with the bait. After controlling positive enrichment ofbinding phages to a bacterially expressed and purified GTS-RhoBL63 inphage ELISA, we wanted to develop a direct screening for RhoBintrabodies. We learned from our previous experiences in recombinantantibody technologies that such monoclonal binding domains efficacy canbe very assay-dependent, namely that positive one in ELISA screen oftenfailed to work in immunofluorescence or vice versa. We also isolatedintrabodies using a selection scheme base on co-localization of afluorescent fusion of the nanobody with the target. Then when wefunctionalized a set of these tracking intrabodies, replacing the GFP bya proteasome targeting domain to degrade the antigen, there wassurprisingly no obvious correlation between the best trackers and thebest degraders. Therefore we reasoned that the best way to identify anintrabody that work in a specific assay would be to screen directly inthe final format.

Here we chose to inhibit RhoB by inducing its proteasome mediateddegradation. Several functionalization of intrabodies mend to inducedegradation of the target. One of them consists in fusing the Fboxdomain of an Fbox protein. Fbox protein contains two modular domains,one for target recognition and the Fbox domain that interact with Skip1,a component of the SCF E3 ubiquitin ligase complex, which inducepolyubiquitinylation of the Fbox protein target followed by subsequentproteasomal degradation. Replacement of the target binding domain withan intrabody can specify the target, therefore inducing degradation ofthe antigen. One advantage of that knockdown strategy is that theFbox-intrabody (F-Ib) act in a catalytic manner and is not co-degraded.Another one resides in the fact that if degradation is observed, thisreport indirectly the intracellular interaction between the antigen andthe nanobody. The main drawback can be that the targeted antigen doesnot display ubiquitinylation site, but it is not the case for smallGTPases or any protein that could be degraded naturally by theproteasome. We have previously tested this strategy for several anti-GFPhs2dAb intrabodies and constructed a plasmid which allows the expressionof an amino terminal Fbox domain from Drosophila slmb gene fused tohs2dAb and a carboxy terminal myc tag upstream of a mitochondrialfluorescent reporter gene expressed as a second cistron translated froman IRES. We choose to set up a visual screen of target degradation byfusing RhoB to a fluorescent protein. To mimic active RhoB, we choose toexpress a constitutively active mutant RhoBL63, which is stronglyimpaired in catalyzing GTP nucleotide hydrolysis, thus remains in theGTP loaded active state. To avoid binding crosstalk with endogenousRhoB, we used a RHOB−/− lung epithelial cell line H2882. As RhoBL63expression toxicity did not allow us to produce a stable cell line, weconstructed a chimera which consist in a sequence coding an aminoterminal histone H2B, followed by the mCherry fluorescent protein and acarboxy terminal RhoBL63 deleted for the 5 terminal amino acid thatcorrespond to the palmitoylation and prenylation signals. This fusionprotein loss the membrane anchorage capacity and was artificiallyincorporated to the chromatin nucleosomes, giving a fluorescent signalin the nucleus while displaying active RhoBL63 mutant at a localizationwhich appeared to be nontoxic to generate a stable cell line, referredas HmB. To control the binding specificity to RhoBL63, a cell lineexpressing only H2B-mCherry was generated as well, referred as Hm. Wehypothesized that if a Fbox-hs2dAb is a stable F-Ib and if interactionoccurs specifically with RhoBL63, a decrease of nuclear mCherryfluorescence would be observed in the HmB cell line but not in the Hmone. Therefore a fluorescence decay correlated to RhoBL63 degradationcould be the basis of a visual screening for F-Ib RhoB inhibitors.Chromatin quantity and density is cell-dependent, fluctuating accordingto the cell cycle, giving a slight heterogeneity in the cell nuclearfluorescence could. Another source of cell-dependent heterogeneity in ascreen based on transient plasmid transfection comes from the variableplasmid copy number, the transfection efficiency and the relativeexpression level of F-Ib. To better assess these parameter, we used ourF-Ib bi-cistronic expression vector with a monomeric GFP targeted to themitochondrial matrix as a reporter gene and set up the assay using twonegative hs2dAb in this screening, referred as F-NR that is non relevantto RhoB phage display and the F-20, previously selected towards RhoB butthat is not a degrading intrabody. In summary, the visual screen residesin the observation of mCherry nuclear fluorescence decay in cellsshowing GFP fluorescent mitochondria.

After 4 rounds of panning, hs2dAb sequence were digested in pool anddirectly inserted in the F-Ib bicistronic vector. Although suchpolyclonal subcloning could lead to a certain extend to diversity losscompared to the phagemid sublibrary, we reasoned that in conventionalphage display strategies, only a set of randomly picked colonies arescreened and that the effective enrichment of specific binders duringphage selection have less probability to be not transferred duringsubcloning. After a single cloning step, we screened several hundred ofF-hs2dAb, by transient transfection of individual plasmid clones in bothcell lines (HmB and Hm), and observed the mCherry fluorescence intensityon an inverted microscope. After sequencing positive hits, we identifiedfour unique clones that induced a strong decay of mCherry fluorescencein HmB cell transfected cells only in comparison with to the twonegative internal controls F-NR and F-B20. One of the selected cloneswas the H12 hs2dAb, which is the pan active Rho that was previouslyidentified from that NaLi-H1 library. The fluorescent decayquantification on some selected field suggested that these F-Ib wereinducing degradation of H2B-mCherry-RhoBL63 depending on the presence ofRhoBL63. Then, these results were further quantified by flow cytometry,confirming that F-H12, F-B6, F-B15 and F-B5 degrade selectivelyH2BmCherry-RhoBL63 and showing that F-H12 and F-B6 are the mostefficient F-Ib (FIG. 3A).

Characterization of Selected F-Ib

The fusion of a Fbox domain to a peptide or an intrabody have beenreported to mediate target degradation by the proteasome in variouscellular context. To confirm whether the presence of the Fbox domain wasresponsible to the degradation, we expressed hs2dAb alone in Hm and HmBcell lines and observed no decrease in mCherry fluorescence (FIG. 3B).Next, using the MG132 proteasome inhibitor, we controlled that theobserved degradation was proteasome dependent. In comparison to DMSOtreatment that barely reduce the 4 F-Ib induced fluorescent decayquantified by flow cytometry, the treatment at 1 μM of MG132 during 36 hrestored the mCherry fluorescence almost to the control level (FIG. 3C).Finally we analyzed whether the fluorescence decay was a direct effectof the F-Ib expression by quantifying the fluorescence after decreasingthe concentration of plasmid in the transfection from 2 μg to 0. Adose-response direct effect was observed for the effective F-H12 andF-B6, as the lower was the plasmid concentration the higher was thefluorescence signal (data not shown). Together these resultsdemonstrated that the F-Ib selected by the direct visual screening werespecifically targeting and degrading in a proteasome dependent mannerthe RhoBL63 delta CAAX protein concentrated on the chromatin.

Specificity and Conformational Selectivity of the Selected F-Ib

The H12 hs2dAb is a conformational sensor and a blocking intrabody ofthe GTP loaded Rho proteins without distinction between RhoA, RhoB, RhoChomologs and even recognizing Rac1 and CDCl42 closely related GTPases.The fact that it was enriched and selected again in this study was notsurprising as in previous panning its enrichment was very high in theearly round of selection as its representation was above 50% of theclones at the third round of panning on RhoAL63. Despite here weintroduced competition with active RhoA and RhoC, H12 was not totallyeliminated from the selection, suggesting that others newly selectedhs2dAb could also be pan Rho as well. Nevertheless H12 enrichment wasmuch lower, suggesting that the new subtractive selection was at leastpartially efficient. To determine the selectivity of the selectedF-hs2dAb, we produced different stable cell lines on the same basis thanH2B-mCherry-RhoBL63. Transfection of H2B-mCherry-RhoAL63 andH2B-mCherry-RhoCL63 failed to produce stable cell line and theheterogeneity of transient expression did not lead to conclusivequantification of fluorescence decay (data not shown). However thegeneration of a similar cell line was possible with H2B-mCherry-Rac1L61,Rac1 being the closest homolog of the Rho subfamily mainly in the switchdomains. As expected, F-H12 induced a fluorescence decay in the latercell line. Among the other selected F-Ib, F-B5 was also affecting thefluorescence level of the H2B-mCherry-Rac1L63 but F-B6 and F-B15 failedto degrade the active form of Rac1 (FIG. 3D). At this point we pursuedthe study without the hs2dAb 5 or its F-5 functionalization but we keptthe hs2dAb H12 as a pan active Rho control. Then we addressed theconformational selectivity of the remaining F-Ib by comparing theireffect on a RhoBN19 mutant which is supposed to be mainly inactive asthe same mutation lead to a GTPase defective in the nucleotide bindingfor other Ras homologs. We generated a H2B-mCherry-RhoBN19 stable cellline in order to determine the conformational selectivity hs2dAbexpressed as F-Ib in our fluorescence decay assay. After FACS analysis,all effective F-Ib were degrading only the active mutant of RhoB and notthe inactive form (FIG. 3E). These results indicate that F-B6 and F-B15are conformational hs2dAb that preferentially recognize RhoB in itsactive conformation.

Endogenous RhoB Activity Knockdown

We then investigated whether these intrabodies were able to degrade theendogenous active form of RhoB. To this end we used HeLa S3 cells, acommon cell line that express significant amount of RhoB protein with adetectable basal level of active RhoB. The standard method to assay theRho GTPase activity is based on a pull down using the GST-RBD. RBD isthe Rho Binding Domain from Rhotekin, a common effector of the three Rhowhich interacts only with the GTP bound Rho. After 48 h of transienttransfection of F-Ib, pull down of RhoB basal active fraction was lowerin cells transfected by F-B6, F-B15 or F-H12 than with the controlsF-B20 and F-NR. Detection of RhoA and RhoC allowed to assess whethertheir basal activities were also affected. As expected F-H12 induced astrong decrease in the level of all 3 Rho active fractions. However, thelevel of the 3 active Rho was not decreased equally for the F-B15 andF-B6 expression, suggesting that they do not have the same selectivitythan F-H12. In contrast to the F-B15 hs2dAb that induced degradation ofboth active RhoB and RhoA, F-B6 did not induced apparent modulation ofRhoA or RhoC pulled down fractions (FIG. 4A). Quantifications indicatedthat F-B6 degrades solely RhoB activity in this cellular context andassay conditions (FIG. 4B). This result is the first example of amolecule which would discriminate RhoB from RhoA in their GTP loadedstate and that would enable their cellular proteolysis.

To investigate whether the protein knockdown observed 48 h posttransfection with the F-6 was direct and specific, we targeted the fastprocess of cellular activation of Rho proteins. Actually RhoB and RhoA,and to a lesser extend RhoC, have been reported to be activated in fewminutes after an EGF treatment. After 24 h of serum starvation,activation kinetics of each Rho by EGF was assessed in HeLaS3 cells.Activation was observed as soon as 5 minutes after stimulation for all 3Rho and reached a maximum at 15 minutes, which was chosen as activationtime for further experiments (FIGS. 4C & 4D). We characterized theeffect of F-H12 and F-B6 on Rho activation and confirmed the selectiveobserved degradation of RhoA/B activity. While F-NR or F-B20 controlsdid not prevent EGF mediated Rho activation, F-B6 degrades only RhoBactivity induced by EGF whereas FH12 inhibit all Rho activities indeed(FIGS. 4C & 4D).

In conclusion, the hs2dAb B6 seems to be a RhoB-GTP very selectiveintrabody, which is able to block RhoB basal activity as well as itsstimulated activation while functionalized as F-Ib, without downregulating major fraction of cellular RhoB.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

We claim:
 1. A nucleic acid molecule which encodes a single domainantibody, wherein the amino acid sequences of CDR1-IMGT, CDR2-IMGT andCDR3-IMGT of the single domain antibody are set forth as SEQ ID NOS:5-7.
 2. A vector which includes the nucleic acid molecule of claim
 1. 3.A host cell transformed with the nucleic acid molecule of claim 1.